Affinity chromatography is chromatography that uses a column packed with a ligand-immobilized carrier, in which a substance (ligand) that specifically binds to a substance intended to be separated or purified is immobilized on an insoluble carrier. Affinity chromatography is used for, for example, separation and purification of bio-related substances such as proteins and nucleic acids (Patent Literature 1). As the carrier for affinity chromatography, for example, crosslinked particles of sugar chains represented by agarose gel, or particles containing a synthetic polymer as a main component are used.
In the production of an affinity chromatography carrier, it is necessary to immobilize a ligand, which is a substance capable of binding specifically to an intended substance, to the carrier. Staphylococcus aureus protein A (SpA) and variants thereof are known as representative affinity chromatography ligands having a binding capacity specific to immunoglobulins. Since SpA has an ability to bind to the Fc region of an immunoglobulin without noticeably affecting the high selectivity of the immunoglobulin for antigens, immunoglobulins and Fc region-containing proteins can be efficiently captured and purified thereby.
Natural-type SpA contains five domains, namely, E, D, A, B and C, in sequence from the N-terminal, as domains having a binding capacity for immunoglobulins. These domains and the Z domain, which is a modified domain of the B domain, are used as the affinity chromatography ligands. Furthermore, for the purpose of increasing the immunoglobulin-binding capacity, it is also common to use a product obtained by linking two or more of the above-mentioned domains together, as a ligand.
It is known that the above-mentioned immunoglobulin-binding domains of SpA respectively contain three α-helix structures, and among these, two α-helix portions on the N-terminal side contribute to the binding to immunoglobulins. Furthermore, in regard to the B and C domains among the above-mentioned domains, it has been reported that a turn structure is formed by Asn at the position 3 and Lys at the position 4 of the N-terminal (Non-Patent Literature 1). Therefore, in regard to a ligand obtained by linking multiple units of the B domain, the C domain, or the Z domain, which is a modified domain of the B domain, it is speculated that the various domains are linked in an arrangement of being crooked from each other due to the turn structures, and the ligand causes steric hindrance attributed to this crooked arrangement. This steric hindrance causes a serious problem in producing an affinity chromatography carrier that employs a repeated structure of the B domain, the C domain or the Z domain as a ligand. That is, the immunoglobulin-binding capacity of the ligand remains low because of the steric hindrance mentioned above, and consequently, a large amount of carrier is required in order to purify a certain amount of immunoglobulins. Affinity chromatography carriers that employ SpA as a ligand are very expensive in many cases, and requiring a large amount of carrier is not desirable from the viewpoint of production cost.
Affinity chromatography carriers are usually used repeatedly in the applications related to bioseparation. Therefore, usually, a cleaning process known as cleaning-in-place (CIP), which is intended for returning a carrier to the original state by eliminating contaminants, is repeatedly carried out during use. Regarding the reagent for the CIP, for example, an alkaline liquid of, e.g., sodium hydroxide is used. However, for those affinity chromatography carriers that use a protein as a ligand, such alkaline conditions are harsh, and the ligands may lose the binding capacity for target molecules as a result of deactivation or cleavage of the ligands. In regard to the deactivation of ligands under such alkaline conditions, deamidation of Asn and Gln residues is widely known as a main cause of the deactivation. Particularly, it has been reported that Asn is highly sensitive to alkaline conditions, and deamidation thereof is structure-dependent and it frequently occurs at an amino acid sequence site represented by Asn-Gly or Asn-Ser (Non-Patent Literature 2).
An example of the prior art technologies for avoiding the deactivation of ligands under alkaline conditions as described above may include obtaining a ligand having decreased sensitivity to alkali as a result of deletion or modification of Asn residues. By using such a ligand, an affinity chromatography carrier that can maintain the immunoglobulin-binding capacity even after a CIP using an alkaline solution has been carried out several times, is provided. For example, in Patent Literature 2, there is provided a ligand for an affinity chromatography carrier, the ligand including the B domain, the C domain or the Z domain of SpA, and the ligand including deletion of at least three consecutive amino acids on the N-terminal side starting from the position 1 or the position 2 of at least one domain. Furthermore, in Patent Literature 3, there is provided a ligand for an affinity chromatography carrier, the ligand including the C domain of SpA in which consecutive amino acids from the position 3 to the position 6 on the N-terminal side have been deleted. In Patent Literature 4, there is provided a ligand for affinity chromatography carrier, the ligand having a modified Asn residue in the Z domain or the B domain of SpA. However, the prior art technologies described above are primarily purported to increase the alkali resistance of carriers, and there is no mention about increasing the immunoglobulin-binding capacity of carriers.